Home › Microbiology › Neutrophil Extracellular Traps: How to Generate and Visualize Them
Microbiology JoVE (Open Access) Citable · DOI

Neutrophil Extracellular Traps: How to Generate and Visualize Them

DOI: 10.3791/1724-v
What you'll learn
  • ✓ Isolate neutrophil granulocytes from human blood using density gradient centrifugation
  • ✓ Activate PMNs to induce neutrophil extracellular trap (NET) formation
  • ✓ Visualize NETs using immunolabeling, light microscopy, and scanning electron microscopy
Protocol

Neutrophil Extracellular Traps (NETs) are an important innate immune mechanism to fight pathogenic bacteria, fungi and parasites. Here we describe methods to isolate neutrophil granulocytes from human blood and to activate them to form NETs. We present preparation techniques to visualize NETs in light and electron microscopy.

Difficulty
intermediate
Total time
~4–6 hours per blood sample (isolation through imaging preparation)
Model organism
Human neutrophils (primary cells from peripheral blood)
Biosafety
BSL-1

Steps

1
Understand neutrophil extracellular trap biology and function

Review the role of NETs in innate immunity against bacteria, fungi, and parasites. Understand the rationale for isolating and activating neutrophils as a model system.

▶ 00:47
2
Isolate polymorphonuclear cells from human blood

Perform density gradient centrifugation on whole blood to separate and collect viable neutrophil granulocytes (PMNs) for downstream activation and analysis.

▶ 01:32
3
Activate isolated PMNs to induce NET formation

Treat isolated neutrophils with activating stimuli (e.g., PMA or bacterial components) to trigger the formation and release of neutrophil extracellular traps.

▶ 05:11
4
Detect NETs using immunolabeling and light microscopy

Fix and immunostain NET samples with fluorescent antibodies against NET-associated proteins to visualize trap structures and quantify NET formation by fluorescence microscopy.

▶ 06:46
5
Prepare NET samples for scanning electron microscopy

Process activated neutrophil cultures through fixation, dehydration, critical-point drying, and metal coating to visualize three-dimensional NET ultrastructure at high resolution.

▶ 08:16
6
Examine representative results and NET morphology

Review light and electron microscopy images showing typical NET morphology, trap dimensions, and DNA-protein architecture from successfully activated samples.

▶ 12:16
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